Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

Rosenthal, Katrin; Becker, Martin; Rolf, Jascha; Siedentop, Regine; Hillen, Michaël; Nett, Markus; Lütz, Stephan

doi:10.1002/cbic.202000433

Cited by 17 publications

(33 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Polyphosphate kinases were found to be suitable for ATP regeneration, as they require inexpensive polyP for the phosphorylation of AMP to ADP and ADP to ATP, respectively. The chosen kinases Sc ADK, Aj PPK2, and Sm PPK2 are a well-established regeneration system of ATP [ 11 ] that was now extended with the enzyme thscGAS, which catalyzes the cyclization of ATP and GTP for 2′3′-cGAMP synthesis [ 24 ].…”

Section: Resultsmentioning

confidence: 99%

“…Descriptions of the plasmids for the expression of Sc ADK, Aj PPK2 and Sm PPK2 can be found in [ 15 ]. The thscGAS expression strain E. coli BL21 (DE3) pLysS pET28a -SUMOthscGAS is described in [ 24 ].…”

Section: Methodsmentioning

confidence: 99%

“…These properties make 2′3′-cGAMP an interesting candidate in drug discovery, e.g., for cancer immunotherapy or as an adjuvant in vaccines [ 21 , 22 ]. The biocatalytic synthesis of 2′3′-cGAMP using cGAS and nucleoside 5′-triphosphates as starting material has already been extensively investigated [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

et al. 2021

Self Cite

View full text Add to dashboard Cite

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2’3’-cGAMP) formation was successfully developed. The 2’3’‑cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2’3’-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2’3’-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The process was designed for an enzyme quantity of 10 g, which is sufficient to synthesize 10 to 1000 g of product corresponding to the amount required for e.g., preclinical or phase 1 clinical studies [ 15 , 16 ]. As reference the recently reported enzyme cGAS was chosen, which has proven to be a promising biocatalyst for the synthesis of cyclic dinucleotides due to its promiscuous properties [ 17 , 18 ]. Cyclic dinucleotides might act as stimulator of interferon genes (STING) agonist and are therefore pursued as strategy for cancer therapy [ 13 ].…”

Section: Resultsmentioning

confidence: 99%

Environmental Assessment of Enzyme Production and Purification

2021

Self Cite

View full text Add to dashboard Cite

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme−1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.

show abstract

“…Large collections of modified 2′3′CDNs analogs were enzymatically prepared from the commercially available ATP and GTP analogs. Most substrate derivatives with modifications at the nucleobase, ribose and the α-thiophosphate were accepted [ 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Design, Synthesis and Biological Evaluation of (2′,5′ and 3′5′-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING)

2020

View full text Add to dashboard Cite

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3′-5′ and 2′-5′ phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2′3′-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (KD = 0.038 μM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (KD = 0.543 μM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.

show abstract

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2′‐3′‐Linked Cyclic Dinucleotides

Cited by 17 publications

References 32 publications

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

Environmental Assessment of Enzyme Production and Purification

Design, Synthesis and Biological Evaluation of (2′,5′ and 3′5′-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING)

Contact Info

Product

Resources

About